Recent studies show that several cell types that mobilize intracellular calcium in response to hormones or neurotransmitters also hydrolyze phosphatidylinositol 4,5- bisphosphate to produce diacylglycerol and inositol trisphosphate (IP3), which may mediate the mobilization of intracellular calcium. In the present study, we investigated the involvement of IP3 in calcium mobilization in rat brain microsomes. Ethanol- induced neurotransmitter release in unstimulated synaptosomes was found to be independent of the extrasynaptosomal calcium concentration. Therefore, the effects of alcohol on microsomal calcium uptake and release were also studied. IP3 caused a rapid release of calcium from brain microsomes. In vitro addition of 100 mM ethanol had no effect on ATP-dependent calcium accumulation in the microsomes, but the same concentration of ethanol released 25% of the total accumulated calcium from the microsomes. Ethanol induced calcium release in a concentration- dependent manner over the range of 30 mM to 500 mM. The amount of calcium release increased with higher alcohols. The effect of ethanol was temperature-dependent, suggesting a diffusion-controlled process for calcium transport. These results indicate that the stimulatory effect of ethanol on resting release of neurotransmitters in rat brain may be due to the microsomal release of calcium.